Streptococcus pneumoniae gene sequence for DNA ligase

ABSTRACT

The invention provides isolated nucleic acid compounds encoding a DNA ligase of  Streptococcus pneumoniae.  Also provided are vectors and transformed host cells for expressing the encoded protein, and a method for identifying compounds that bind and/or inhibit said protein.

This application claims the benefit of U.S. provisional application No. 60/036,281, filed Dec. 13, 1996.

BACKGROUND OF THE INVENTION

This invention provides isolated DNA sequences, proteins encoded thereby, and methods of using said DNA and protein in a variety of applications.

Widespread antibiotic resistance in common pathogenic bacterial species has justifiably alarmed the medical and research communities. Frequently, resistant organisms are co-resistant to several antibacterial agents. Penicillin resistance in Streptococcus pneumoniae has been particularly problematic. This organism causes upper respiratory tract infections. Modification of a penicillin-binding protein (PBP) underlies resistance to penicillin in the majority of cases. Combating resistance to antibiotic agents will require research into the molecular biology of pathogenic organisms. The goal of such research will be to identify new antibacterial agents.

While researchers continue to develop antibiotics effective against a number of microorganisms, Streptococcus pneumoniae has been more refractory. In part, this is because Streptococcus pneumoniae is highly recombinogenic and readily takes up exogenous DNA from its surroundings. Thus, there is a need for new antibacterial compounds and new targets for antibacterial therapy in Streptococcus pneumoniae.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an isolated gene and encoded protein from S. pneumoniae. The invention enables: (1) preparation of probes and primers for use in hybridizations and PCR amplifications, (2) production of proteins and RNAs encoded by said gene and related nucleic acids, and (3) methods to identify compounds that bind and/or inhibit said protein(s).

In one embodiment the present invention relates to an isolated nucleic acid molecule encoding a DNA ligase protein.

In another embodiment, the invention relates to a nucleic acid molecule comprising the nucleotide sequence identified as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In another embodiment, the present invention relates to a nucleic acid that encodes SEQ ID NO:2.

In another embodiment the present invention relates to an isolated protein molecule, wherein said protein molecule comprises the sequence identified as SEQ ID NO:2.

In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates the Ligase gene (viz. “Lig”) in operable linkage to gene expression sequences enabling the gene to be transcribed and translated in a host cell.

In still another embodiment the present invention relates to host cells that have been transformed or transfected with the cloned Lig gene such that the Lig gene is expressed in the host cell.

This invention also provides a method of determining whether a nucleic acid sequence of the present invention, or fragment thereof, is present in a sample, comprising contacting the sample, under suitable hybridization conditions, with a nucleic acid probe of the present invention.

In a still further embodiment, the present invention relates to a method for identifying compounds that bind and/or inhibit the Lig protein.

DETAILED DESCRIPTION OF THE INVENTION

“ORF” (i.e. “open reading frame”) designates a region of genomic DNA beginning with a Met or other initiation codon and terminating with a translation stop codon, that potentially encodes a protein product. “Partial ORF” means a portion of an ORF as disclosed herein such that the initiation codon, the stop codon, or both are not disclosed.

“Consensus sequence” refers to an amino acid or nucleotide sequence that may suggest the biological function of a protein, DNA, or RNA molecule. Consensus sequences are identified by comparing proteins, RNAs, and gene homologues from different species.

The terms “cleavage” or “restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (viz. sequence-specific endonucleases). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements are used in the manner well known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can readily be found in the literature.

“Essential genes” or “essential ORFs” or “essential proteins” refer to genomic information or the protein(s) or RNAs encoded thereby, that when disrupted by knockout mutation, or by other mutation, result in a loss of viability of cells harboring said mutation.

“Non-essential genes” or “non-essential ORFs” or “non-essential proteins” refer to genomic information or the protein(s) or RNAs encoded therefrom which when disrupted by knockout mutation, or other mutation, do not result in a loss of viability of cells harboring said mutation.

“Minimal gene set” refers to a genus comprising about 256 genes conserved among different bacteria such as M. genitalium and H. influenzae. The minimal gene set may be necessary and sufficient to sustain life. See e.g. A. Mushegian and E. Koonin, “A minimal gene set for cellular life derived by comparison of complete bacterial genomes” Proc. Nat. Acad. Sci. 93, 10268-273 (1996).

“Knockout mutant” or “knockout mutation” as used herein refers to an in vitro engineered disruption of a region of native chromosomal DNA, typically within a protein coding region, such that a foreign piece of DNA is inserted within the native sequence. A knockout mutation occurring in a protein coding region prevents expression of the wild-type protein. This usually leads to loss of the function provided by the protein. A “knockout cassette” refers to a fragment of native chromosomal DNA having cloned therein a foreign piece of DNA that may provide a selectable marker.

The term “plasmid” refers to an extrachromosomal genetic element. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

“Recombinant DNA cloning vector” as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.

The term “recombinant DNA expression vector” as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present to enable transcription of the inserted DNA.

The term “vector” as used herein refers to a nucleic acid compound used for introducing exogenous DNA into host cells. A vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.

The terms “complementary” or “complementarity” as used herein refer to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding to form double stranded nucleic acid molecules. The following base pairs are related by complementarity: guanine and cytosine; adenine and thymine; and adenine and uracil. As used herein, “complementary” applies to all base pairs comprising two single-stranded nucleic acid molecules. “Partially complementary” means one of two single-stranded nucleic acid molecules is shorter than the other, such that one of the molecules remains partially single-stranded.

“Oligonucleotide” refers to a short nucleotide chain comprising from about 2 to about 25 nucleotides.

“Isolated nucleic acid compound” refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.

A “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule.

The term “promoter” refers to a DNA sequence which directs transcription of DNA to RNA.

A “probe” as used herein is a labeled nucleic acid compound which can be used to hybridize with another nucleic acid compound.

The term “hybridization” or “hybridize” as used herein refers to the process by which a single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing.

“Substantially purified” as used herein means a specific isolated nucleic acid or protein, or fragment thereof, in which substantially all contaminants (i.e. substances that differ from said specific molecule) have been separated from said nucleic acid or protein. For example, a protein may, but not necessarily, be “substantially purified” by the IMAC method as described herein.

“Selective hybridization” refers to hybridization under conditions of high stringency. The degree of hybridization between nucleic acid molecules depends upon, for example, the degree of complementarity, the stringency of hybridization, and the length of hybridizing strands.

The term “stringency” relates to nucleic acid hybridization conditions. High stringency conditions disfavor non-homologous base pairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by changes in temperature and salt concentration. Typical high stringency conditions comprise hybridizing at 50° C. to 65° C. in 5X SSPE and 50% formamide, and washing at 50° C. to 65° C. in 0.5X SSPE; typical low stringency conditions comprise hybridizing at 35° C. to 37° C. in 5X SSPE and 40% to 45% formamide and washing at 42° C. in 1X-2X SSPE.

“SSPE” denotes a hybridization and wash solution comprising sodium chloride, sodium phosphate, and EDTA, at pH 7.4. A 20X solution of SSPE is made by dissolving 174 g of NaCl, 27.6 g of NaH2PO4.H2O, and 7.4 g of EDTA in 800 ml of H2O. The pH is adjusted with NaOH and the volume brought to 1 liter.

“SSC” denotes a hybridization and wash solution comprising sodium chloride and sodium citrate at pH 7. A 20X solution of SSC is made by dissolving 175 g of NaCl and 88 g of sodium citrate in 800 ml of H2. The volume is brought to 1 liter after adjusting the pH with 10N NaOH.

DETAILED DESCRIPTION OF THE INVENTION

The Lig gene disclosed herein (SEQ ID NO:1) and related nucleic acids encode a DNA ligase from S. pneumoniae that is essential for viability (SEQ ID NO:2). This gene and protein are members of the minimal gene set. The proteins categorized as “minimal gene set” counterparts are homologous to a set of highly conserved proteins found in other bacteria. The minimal gene set proteins are thought to be essential for viability and are useful targets for the development of new antibacterial compounds.

In one embodiment, the proteins of this invention are purified, and used in a screen to identify compounds that bind and/or inhibit the activity of said proteins. A variety of suitable screens are contemplated for this purpose. For example, the protein(s) can be labeled by known techniques, such as radiolabeling or fluorescent tagging, or by labeling with biotin/avidin. Thereafter, binding of a test compound to a labeled protein can be determined by any suitable means, well known to the skilled artisan.

Skilled artisans will recognize that the DNA molecules of this invention, or fragments thereof, or complements thereof, can be generated by general cloning methods. PCR amplification using oligonucleotide primers targeted to any suitable region of SEQ ID NO:l, SEQ ID NO:4, or SEQ ID NO:5 is preferred. Methods for PCR amplification are widely known in the art. See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990) or U.S. Pat. No. 4,889,818, which hereby is incorporated by reference. A PCR comprises DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, Conn.). A positive PCR result is determined by, for example, detecting an appropriately-sized DNA fragment following agarose gel electrophoresis.

The DNAs of the present invention may also be produced using synthetic methods well known in the art. (See, e.g., E. L. Brown, R. Belagaje, M. J. Ryan, and H. G. Khorana, Methods in Enzymology, 68:109-151 (1979)). An apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404) may be used to synthesize DNA. Synthetic methods rely upon phosphotriester chemistry [See, e.g., M. J. Gait, ed., Oligonucleotide Synthesis, A Practical Approach, (1984)], or phosphoramidite chemistry.

Protein Production Methods

The present invention relates further to substantially purified proteins encoded by the gene disclosed herein.

Skilled artisans will recognize that proteins can be synthesized by different methods, for example, chemical methods or recombinant methods, as described in U.S. Pat. No. 4,617,149, which hereby is incorporated by reference.

The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts relating to this area. See, e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981) Springer-Verlag, New York, 54-92. Peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) and synthesis cycles supplied by Applied Biosystems. Protected amino acids, such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.

The proteins of the present invention can also be made by recombinant DNA methods. Recombinant methods are preferred if a high yield is desired. Recombinant methods involve expressing the cloned gene in a suitable host cell. The gene is introduced into the host cell by any suitable means, well known to those skilled in the art. While chromosomal integration of the cloned gene is within the scope of the present invention, it is preferred that the cloned gene be maintained extra-chromosomally, as part of a vector in which the gene is in operable-linkage to a promoter.

Recombinant methods can also be used to overproduce a membrane-bound or membrane-associated protein. In some cases, membranes prepared from recombinant cells expressing such proteins provide an enriched source of the protein.

Expressing Recombinant Proteins in Procaryotic and Eucaryotic Host Cells

Procaryotes are generally used for cloning DNA sequences and for constructing vectors. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for expression of foreign proteins. Other strains of E. coli, bacilli such as Bacillus subtilis, enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, various Pseudomonas species may also be employed as host cells in cloning and expressing the recombinant proteins of this invention. Also contemplated are various strains of Streptococcus and Streptocmyces.

For effective recombinant protein production, a gene must be linked to a promoter sequence. Suitable bacterial promoters include b -lactamase [e.g. vector pGX2907, ATCC 39344, contains a replicon and b -lactamase gene], lactose systems [Chang et al., Nature (London), 275:615 (1978); Goeddel et al., Nature (London), 281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promoter system [vector pATH1 (ATCC 37695)] designed for the expression of a trpE fusion protein. Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540, ATCC-37282) are also suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence, operably linked to the DNA encoding the desired polypeptides. These examples are illustrative rather than limiting.

A variety of mammalian cells and yeasts are also suitable hosts. The yeast Saccharomyces cerevisiae is commonly used. Other yeasts, such as Kluyveromyces lactis, are also suitable. For expression of recombinant genes in Saccharomyces, the plasmid YRp7 (ATCC-40053), for example, may be used. See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman et al., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980). Plasmid YRp7 contains the TRP1 gene, a selectable marker for a trpl mutant.

Purification of Recombinantly-Produced Protein

An expression vector carrying a nucleic acid or gene of the present invention is transformed or transfected into a suitable host cell using standard methods. Cells that contain the vector are propagated under conditions suitable for expression of a recombinant protein. For example, if the gene is under the control of an inducible promoter, then suitable growth conditions would incorporate the appropriate inducer. The recombinantly-produced protein may be purified from cellular extracts of transformed cells by any suitable means.

In a preferred process for protein purification a gene is modified at the 5′ end, or at some other position, such that the encoded protein incorporates several histidine residues (viz. “histidine tag”). This “histidine tag” enables “immobilized metal ion affinity chromatography” (IMAC), a single-step protein purification method described in U.S. Pat. No. 4,569,794, which hereby is incorporated by reference. The IMAC method enables isolation of substantially pure protein starting from a crude cellular extract.

As skilled artisans will recognize, owing to the degeneracy of the code, the proteins of the invention can be encoded by a large genus of different nucleic acid sequences. This invention further comprises said genus.

The ribonucleic acid compounds of the invention may be prepared using the polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically using RNA polymerase to transcribe a DNA template.

The most preferred systems for preparing the ribonucleic acids of the present invention employ the RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5′ end of a template. See, J. Sambrook, et al., supra, at 18.82-18.84.

This invention also provides nucleic acids that are complementary to the sequences disclosed herein.

The present invention also provides probes and primers, useful for a variety of molecular biology techniques including, for example, hybridization screens of genomic or subgenomic libraries, or detection and quantification of MRNA species as a means to analyze gene expression. A nucleic acid compound is provided comprising any of the sequences disclosed herein, or a complementary sequence thereof, or a fragment thereof, which is at least 15 base pairs in length, and which will hybridize selectively to Streptococcus pneumoniae DNA or mRNA. Preferably, the 15 or more base pair compound is DNA. A probe or primer length of at least 15 base pairs is dictated by theoretical and practical considerations. See e.g. B. Wallace and G. Miyada, “Oligonucleotide Probes for the Screening of Recombinant DNA Libraries,” In Methods in Enzymology, Vol. 152, 432-442, Academic Press (1987).

The probes and primers of this invention can be prepared by methods well known to those skilled in the art (See e.g. Sambrook et al. supra). In a preferred embodiment the probes and primers are synthesized by the polymerase chain reaction (PCR).

The present invention also relates to recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention. Preferred nucleic acid vectors are those that comprise DNA. The skilled artisan understands that choosing the most appropriate cloning vector or expression vector depends on the availability of restriction sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into a host chromosome), the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another), and the number of gene copies desired in the host cell.

Suitable vectors comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like. The most preferred vectors are plasmids.

Host cells harboring the nucleic acids disclosed herein are also provided by the present invention. A preferred host is E. coli transfected or transformed with a vector comprising a nucleic acid of the present invention.

The invention also provides a host cell capable of expressing a gene described herein, said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector comprising an isolated DNA sequence that encodes said gene. The preferred host cell is any strain of E. coli that can accommodate high level expression of an exogenously introduced gene. Transformed host cells are cultured under conditions well known to skilled artisans, such that said gene is expressed, thereby producing the encoded protein in the recombinant host cell.

To discover compounds having antibacterial activity, one can look for agents that inhibit cell growth and/or viability by, for example, inhibiting enzymes required for cell wall biosynthesis, and/or by identifying agents that interact with membrane proteins. A method for identifying such compounds comprises contacting a suitable protein or membrane preparation with a test compound and monitoring by any suitable means an interaction and/or inhibition of a protein of this invention.

For example, the instant invention provides a screen for compounds that interact with the proteins of the invention, said screen comprising:

a) preparing a protein, or membranes enriched in a protein;

b) exposing the protein or membranes to a test compound; and

c) detecting an interaction of a protein with said compound by any suitable means.

The screening method of this invention may be adapted to automated procedures such as a PANDEX® (Baxter-Dade Diagnostics) system, allowing for efficient high-volume screening of compounds.

In a typical screen, a protein is prepared as described herein, preferably using recombinant DNA technology. A test compound is introduced into a reaction vessel containing said protein. The reaction/interaction of said protein and said compound is monitored by any suitable means. In a preferred method, a radioactively-labeled or chemically-labeled compound or protein is used. A specific association between the test compound and protein is monitored by any suitable means.

In such a screening protocol Lig is prepared as described herein, preferably using recombinant DNA technology. A test compound is introduced into a reaction vessel containing the Lig protein or fragment thereof. Binding of Lig by a test compound is determined by any suitable means. For example, in one method radioactively-labeled or chemically-labeled test compound may be used. Binding of the protein by the compound is assessed, for example, by quantifying bound label versus unbound label using any suitable method. Binding of a test compound may also be carried out by a method disclosed in U.S. Pat. No. 5,585,277, which hereby is incorporated by reference. In this method, binding of a test compound to a protein is assessed by monitoring the ratio of folded protein to unfolded protein, for example by monitoring sensitivity of said protein to a protease, or amenability to binding of said protein by a specific antibody against the folded state of the protein.

The foregoing screening methods are useful for identifying a ligand of a Lig protein, perhaps as a lead to a pharmaceutical compound for modulating the state of differentiation of an appropriate tissue. A ligand that binds Lig, or related fragment thereof, is identified, for example, by combining a test ligand with Lig under conditions that cause the protein to exist in a ratio of folded to unfolded states. If the test ligand binds the folded state of the protein, the relative amount of folded protein will be higher than in the case of a test ligand that does not bind the protein. The ratio of protein in the folded versus unfolded state is easily determinable by, for example, susceptibility to digestion by a protease, or binding to a specific antibody, or binding to chaperonin protein, or binding to any suitable surface.

The following examples more fully describe the present invention. Those skilled in the art will recognize that the particular reagents, equipment, and procedures described are merely illustrative and are not intended to limit the present invention in any manner.

EXAMPLE 1 Production of a Vector for Expressing S. pneumoniae Lig in a Host Cell

An expression vector suitable for expressing is. pneumoniae Lig in a variety of procaryotic host cells, such as E. coli, is easily made. The vector contains an origin of replication (Ori), an ampicillin resistance gene (Amp) useful for selecting cells which have incorporated the vector following a tranformation procedure, and further comprises the T7 promoter and T7 terminator sequences in operable linkage to the Lig coding region. Plasmid pET11A (obtained from Novogen, Madison, Wis.) is a suitable parent plasmid. pET11A is linearized by restriction with endonucleases NdeI and BamHI. Linearized pET11A is ligated to a DNA fragment bearing NdeI and BamHI sticky ends and comprising the coding region of the S. pneumoniae Lig (SEQ ID NO:1). The coding region for Lig is easily produced by PCR technology using suitably designed primers to the ends of the coding region specified in SEQ ID NO:1.

The Lig gene used in this construction is slightly modified at the 5′ end (amino terminus of encoded protein) in order to simplify purification of the encoded protein product. For this purpose, an oligonucleotide encoding 8 histidine residues is inserted after the ATG start codon. Placement of the histidine residues at the amino terminus of the encoded protein serves to enable the IMAC one-step protein purification procedure.

EXAMPLE 2 Recombinant Expression and Purification of a Protein Encoded by S. pneumoniae Lig

An expression vector that carries Lig from the S. pneumoniae genome as disclosed herein and which Lig is operably-linked to an expression promoter is transformed into E. coli BL21 (DE3)(hsdS gal lcIts857 ind1Sam7nin5lacUV5-T7gene 1) using standard methods (see Example 4). Transformants, selected for resistance to ampicillin, are chosen at random and tested for the presence of the vector by agarose gel electrophoresis using quick plasmid preparations. Colonies which contain the vector are grown in L broth and the protein product encoded by the vector-borne ORF is purified by immobilized metal ion affinity chromatography (IMAC), essentially as described in U.S. Pat. No. 4,569,794.

Briefly, the IMAC column is prepared as follows. A metal-free chelating resin (e.g. Sepharose 6B IDA, Pharmacia) is washed in distilled water to remove preservative substances and infused with a suitable metal ion [e.g. Ni(II), Co(II), or Cu(II)] by adding a 50mM metal chloride or metal sulfate aqueous solution until about 75% of the interstitial spaces of the resin are saturated with colored metal ion. The column is then ready to receive a crude cellular extract containing the recombinant protein product.

After removing unbound proteins and other materials by washing the column with any suitable buffer, pH 7.5, the bound protein is eluted in any suitable buffer at pH 4.3, or preferably with an imidizole-containing buffer at pH 7.5.

5 1959 base pairs nucleic acid single linear DNA (genomic) NO NO CDS 1..1959 1 ATG AAT AAA AGA ATG AAT GAG TTA GTC GCT TTG CTC AAT CGC TAT GCG 48 Met Asn Lys Arg Met Asn Glu Leu Val Ala Leu Leu Asn Arg Tyr Ala 1 5 10 15 ACT GAG TAC TAT ACC AGC GAT AAT CCC TCG GTT TCA GAC AGT GAG TAT 96 Thr Glu Tyr Tyr Thr Ser Asp Asn Pro Ser Val Ser Asp Ser Glu Tyr 20 25 30 GAC CGC CTT TAC CGT GAG TTG GTC GAG TTA GAA ACT GCT TAT CCA GAG 144 Asp Arg Leu Tyr Arg Glu Leu Val Glu Leu Glu Thr Ala Tyr Pro Glu 35 40 45 CAA GTG CTA GCA GAC AGT CCG ACT CAT CGT GTT GGT GGC AAG GTT TTA 192 Gln Val Leu Ala Asp Ser Pro Thr His Arg Val Gly Gly Lys Val Leu 50 55 60 GAT GGT TTT GAA AAA TAC AGT CAT CAG TAT CCT CTT TAT AGT TTG CAG 240 Asp Gly Phe Glu Lys Tyr Ser His Gln Tyr Pro Leu Tyr Ser Leu Gln 65 70 75 80 GAT GCT TTT TCA CGT GAG GAG CTA GAT GCT TTT GAT GCG CGT GTT CGT 288 Asp Ala Phe Ser Arg Glu Glu Leu Asp Ala Phe Asp Ala Arg Val Arg 85 90 95 AAG GAA GTG GCT CAT CCG ACC TAT ATT TGT GAG CTG AAA ATC GAT GGC 336 Lys Glu Val Ala His Pro Thr Tyr Ile Cys Glu Leu Lys Ile Asp Gly 100 105 110 TTA TCT ATC TCG CTG ACT TAT GAA AAG GGG ATT TTG GTT GCT GGG GTA 384 Leu Ser Ile Ser Leu Thr Tyr Glu Lys Gly Ile Leu Val Ala Gly Val 115 120 125 ACA CGT GGA GAT GGT TCA ATT GGT GAA AAT ATC ACA GAA AAC CTC AAG 432 Thr Arg Gly Asp Gly Ser Ile Gly Glu Asn Ile Thr Glu Asn Leu Lys 130 135 140 CGT GTT AAG GAC ATC CCT TTG ACT TTG CCA GAA GAA CTA GAT ATC ACA 480 Arg Val Lys Asp Ile Pro Leu Thr Leu Pro Glu Glu Leu Asp Ile Thr 145 150 155 160 GTT CGT GGG GAA TGT TAC ATG CCA CGC GCT TCC TTT GAC CAA GTT AAC 528 Val Arg Gly Glu Cys Tyr Met Pro Arg Ala Ser Phe Asp Gln Val Asn 165 170 175 CAA GCG CGC CAA GAA AAT GGA GAG CCT GAA TTT GCT AAT CCT CGT AAT 576 Gln Ala Arg Gln Glu Asn Gly Glu Pro Glu Phe Ala Asn Pro Arg Asn 180 185 190 GCG GCA GCA GGA ACT CTG CGT CAG TTG GAT ACA GCA GTA GTT GCC AAG 624 Ala Ala Ala Gly Thr Leu Arg Gln Leu Asp Thr Ala Val Val Ala Lys 195 200 205 CGT AAT CTT GCA ACG TTT CTC TAT CAA GAA GCC AGC CCT TCA ACT CGT 672 Arg Asn Leu Ala Thr Phe Leu Tyr Gln Glu Ala Ser Pro Ser Thr Arg 210 215 220 GAT AGC CAA GAA AAG GGT TTG AAG TAC CTA GAA CAA CTA GGT TTT GTG 720 Asp Ser Gln Glu Lys Gly Leu Lys Tyr Leu Glu Gln Leu Gly Phe Val 225 230 235 240 GTC AAT CCT AAG CGA ATC TTG GCT GAA AAC ATA GAT GAA ATC TGG AAT 768 Val Asn Pro Lys Arg Ile Leu Ala Glu Asn Ile Asp Glu Ile Trp Asn 245 250 255 TTT ATC CAA GAA GTA GGA CAG GAA CGG GAA AAT CTG CCT TAC GAT ATT 816 Phe Ile Gln Glu Val Gly Gln Glu Arg Glu Asn Leu Pro Tyr Asp Ile 260 265 270 GAT GGA GTG GTA ATC AAG GTC AAC GAC CTA GCA AGT CAA GAA GAA CTT 864 Asp Gly Val Val Ile Lys Val Asn Asp Leu Ala Ser Gln Glu Glu Leu 275 280 285 GGT TTT ACC GTT AAG GCT CCA AAG TGG GCA GTA GCC TAC AAG TTC CCT 912 Gly Phe Thr Val Lys Ala Pro Lys Trp Ala Val Ala Tyr Lys Phe Pro 290 295 300 GCT GAA GAA AAA GAA GCT CAA CTC TTA TCA GTT GAC TGG ACA GTT GGC 960 Ala Glu Glu Lys Glu Ala Gln Leu Leu Ser Val Asp Trp Thr Val Gly 305 310 315 320 CGT ACC GGT GTT GTA ACT CCA ACT GCT AAT CTA ACA CCA GTA CAA CTT 1008 Arg Thr Gly Val Val Thr Pro Thr Ala Asn Leu Thr Pro Val Gln Leu 325 330 335 GCC GGT ACG ACT GTT AGC CGT GCG ACC CTG CAC AAT GTA GAT TAT ATT 1056 Ala Gly Thr Thr Val Ser Arg Ala Thr Leu His Asn Val Asp Tyr Ile 340 345 350 GCT GAA AAA GAT ATC CGA AAA GAC GAT ACG GTC ATT GTA TAT AAG GCT 1104 Ala Glu Lys Asp Ile Arg Lys Asp Asp Thr Val Ile Val Tyr Lys Ala 355 360 365 GGT GAC ATC ATC CCT GCC GTT TTA CGT GTG GTA GAG TCC AAA CGG GTT 1152 Gly Asp Ile Ile Pro Ala Val Leu Arg Val Val Glu Ser Lys Arg Val 370 375 380 TCT GAA GAA AAA CTA GAT ATC CCT ACA AAC TGT CCA AGT TGT AAC TCT 1200 Ser Glu Glu Lys Leu Asp Ile Pro Thr Asn Cys Pro Ser Cys Asn Ser 385 390 395 400 GAC TTG TTG CAC TTT GAA GAT GAA GTG GCC CTA CGT TGT ATC AAT CCG 1248 Asp Leu Leu His Phe Glu Asp Glu Val Ala Leu Arg Cys Ile Asn Pro 405 410 415 CGT TGC CCT GCT CAA ATC ATG GAA GGC TTG ATT CAC TTT GCT TCT CGT 1296 Arg Cys Pro Ala Gln Ile Met Glu Gly Leu Ile His Phe Ala Ser Arg 420 425 430 GAT GCT ATG AAT ATT ACA GGC CTT GGT CCA TCT ATT GTT GAG AAG CTT 1344 Asp Ala Met Asn Ile Thr Gly Leu Gly Pro Ser Ile Val Glu Lys Leu 435 440 445 TTT GCT GCT AAT TTA GTC AAG GAT GTG GCG GAT ATT TAT CGT TTG CAA 1392 Phe Ala Ala Asn Leu Val Lys Asp Val Ala Asp Ile Tyr Arg Leu Gln 450 455 460 GAA GAG GAT TTC CTC CTT TTA GAG GGG GTT AAG GAA AAG TCC GCT GCT 1440 Glu Glu Asp Phe Leu Leu Leu Glu Gly Val Lys Glu Lys Ser Ala Ala 465 470 475 480 AAA CTG TAT CAG GCT ATC CAA GCA TCA AAG GAA AAT TCT GCC GAG AAG 1488 Lys Leu Tyr Gln Ala Ile Gln Ala Ser Lys Glu Asn Ser Ala Glu Lys 485 490 495 CTC TTA TTT GGT TTG GGA ATT CGT CAT GTC GGA AGC AAG GCT AGT CAG 1536 Leu Leu Phe Gly Leu Gly Ile Arg His Val Gly Ser Lys Ala Ser Gln 500 505 510 CTT TTA CTT CAA TAT TTC CAT TCA ATT GAA AAT CTG TAT CAG GCA GAT 1584 Leu Leu Leu Gln Tyr Phe His Ser Ile Glu Asn Leu Tyr Gln Ala Asp 515 520 525 TCA GAG GAA GTG GCT AGT ATT GAA AGT CTA GGT GGC GTG ATT GCC AAA 1632 Ser Glu Glu Val Ala Ser Ile Glu Ser Leu Gly Gly Val Ile Ala Lys 530 535 540 AGT CTT CAG ACT TAT TTT GCG GCA GAA GGC TCT GAA ATT CTG CTC AGA 1680 Ser Leu Gln Thr Tyr Phe Ala Ala Glu Gly Ser Glu Ile Leu Leu Arg 545 550 555 560 GAA TTG AAA GAA ACT GGG GTC AAT CTG GAC TAT AAA GGA CAG ACG GTA 1728 Glu Leu Lys Glu Thr Gly Val Asn Leu Asp Tyr Lys Gly Gln Thr Val 565 570 575 GTA GCG GAT GCG GCC TTG TCA GGT TTG ACC GTG GTA TTG ACA GGA AAA 1776 Val Ala Asp Ala Ala Leu Ser Gly Leu Thr Val Val Leu Thr Gly Lys 580 585 590 TTG GAA CGA CTC AAG CGC TCA GAA GCT AAA AGT AAA CTC GAA AGT CTG 1824 Leu Glu Arg Leu Lys Arg Ser Glu Ala Lys Ser Lys Leu Glu Ser Leu 595 600 605 GGT GCC AAA GTG ACA GGT AGT GTT TCT AAA AAG ACC GAC CTC GTC GTG 1872 Gly Ala Lys Val Thr Gly Ser Val Ser Lys Lys Thr Asp Leu Val Val 610 615 620 GTA GGT GCA GAC GCT GGA AGT AAA CTG CAA AAA GCA CAA GAA CTT GGT 1920 Val Gly Ala Asp Ala Gly Ser Lys Leu Gln Lys Ala Gln Glu Leu Gly 625 630 635 640 ATC CAG GTC AGA GAT GAG GCA TGG CTA GAA AGT TTG TAA 1959 Ile Gln Val Arg Asp Glu Ala Trp Leu Glu Ser Leu * 645 650 652 amino acids amino acid linear protein 2 Met Asn Lys Arg Met Asn Glu Leu Val Ala Leu Leu Asn Arg Tyr Ala 1 5 10 15 Thr Glu Tyr Tyr Thr Ser Asp Asn Pro Ser Val Ser Asp Ser Glu Tyr 20 25 30 Asp Arg Leu Tyr Arg Glu Leu Val Glu Leu Glu Thr Ala Tyr Pro Glu 35 40 45 Gln Val Leu Ala Asp Ser Pro Thr His Arg Val Gly Gly Lys Val Leu 50 55 60 Asp Gly Phe Glu Lys Tyr Ser His Gln Tyr Pro Leu Tyr Ser Leu Gln 65 70 75 80 Asp Ala Phe Ser Arg Glu Glu Leu Asp Ala Phe Asp Ala Arg Val Arg 85 90 95 Lys Glu Val Ala His Pro Thr Tyr Ile Cys Glu Leu Lys Ile Asp Gly 100 105 110 Leu Ser Ile Ser Leu Thr Tyr Glu Lys Gly Ile Leu Val Ala Gly Val 115 120 125 Thr Arg Gly Asp Gly Ser Ile Gly Glu Asn Ile Thr Glu Asn Leu Lys 130 135 140 Arg Val Lys Asp Ile Pro Leu Thr Leu Pro Glu Glu Leu Asp Ile Thr 145 150 155 160 Val Arg Gly Glu Cys Tyr Met Pro Arg Ala Ser Phe Asp Gln Val Asn 165 170 175 Gln Ala Arg Gln Glu Asn Gly Glu Pro Glu Phe Ala Asn Pro Arg Asn 180 185 190 Ala Ala Ala Gly Thr Leu Arg Gln Leu Asp Thr Ala Val Val Ala Lys 195 200 205 Arg Asn Leu Ala Thr Phe Leu Tyr Gln Glu Ala Ser Pro Ser Thr Arg 210 215 220 Asp Ser Gln Glu Lys Gly Leu Lys Tyr Leu Glu Gln Leu Gly Phe Val 225 230 235 240 Val Asn Pro Lys Arg Ile Leu Ala Glu Asn Ile Asp Glu Ile Trp Asn 245 250 255 Phe Ile Gln Glu Val Gly Gln Glu Arg Glu Asn Leu Pro Tyr Asp Ile 260 265 270 Asp Gly Val Val Ile Lys Val Asn Asp Leu Ala Ser Gln Glu Glu Leu 275 280 285 Gly Phe Thr Val Lys Ala Pro Lys Trp Ala Val Ala Tyr Lys Phe Pro 290 295 300 Ala Glu Glu Lys Glu Ala Gln Leu Leu Ser Val Asp Trp Thr Val Gly 305 310 315 320 Arg Thr Gly Val Val Thr Pro Thr Ala Asn Leu Thr Pro Val Gln Leu 325 330 335 Ala Gly Thr Thr Val Ser Arg Ala Thr Leu His Asn Val Asp Tyr Ile 340 345 350 Ala Glu Lys Asp Ile Arg Lys Asp Asp Thr Val Ile Val Tyr Lys Ala 355 360 365 Gly Asp Ile Ile Pro Ala Val Leu Arg Val Val Glu Ser Lys Arg Val 370 375 380 Ser Glu Glu Lys Leu Asp Ile Pro Thr Asn Cys Pro Ser Cys Asn Ser 385 390 395 400 Asp Leu Leu His Phe Glu Asp Glu Val Ala Leu Arg Cys Ile Asn Pro 405 410 415 Arg Cys Pro Ala Gln Ile Met Glu Gly Leu Ile His Phe Ala Ser Arg 420 425 430 Asp Ala Met Asn Ile Thr Gly Leu Gly Pro Ser Ile Val Glu Lys Leu 435 440 445 Phe Ala Ala Asn Leu Val Lys Asp Val Ala Asp Ile Tyr Arg Leu Gln 450 455 460 Glu Glu Asp Phe Leu Leu Leu Glu Gly Val Lys Glu Lys Ser Ala Ala 465 470 475 480 Lys Leu Tyr Gln Ala Ile Gln Ala Ser Lys Glu Asn Ser Ala Glu Lys 485 490 495 Leu Leu Phe Gly Leu Gly Ile Arg His Val Gly Ser Lys Ala Ser Gln 500 505 510 Leu Leu Leu Gln Tyr Phe His Ser Ile Glu Asn Leu Tyr Gln Ala Asp 515 520 525 Ser Glu Glu Val Ala Ser Ile Glu Ser Leu Gly Gly Val Ile Ala Lys 530 535 540 Ser Leu Gln Thr Tyr Phe Ala Ala Glu Gly Ser Glu Ile Leu Leu Arg 545 550 555 560 Glu Leu Lys Glu Thr Gly Val Asn Leu Asp Tyr Lys Gly Gln Thr Val 565 570 575 Val Ala Asp Ala Ala Leu Ser Gly Leu Thr Val Val Leu Thr Gly Lys 580 585 590 Leu Glu Arg Leu Lys Arg Ser Glu Ala Lys Ser Lys Leu Glu Ser Leu 595 600 605 Gly Ala Lys Val Thr Gly Ser Val Ser Lys Lys Thr Asp Leu Val Val 610 615 620 Val Gly Ala Asp Ala Gly Ser Lys Leu Gln Lys Ala Gln Glu Leu Gly 625 630 635 640 Ile Gln Val Arg Asp Glu Ala Trp Leu Glu Ser Leu 645 650 1959 base pairs nucleic acid single linear mRNA NO NO 3 AUGAAUAAAA GAAUGAAUGA GUUAGUCGCU UUGCUCAAUC GCUAUGCGAC UGAGUACUAU 60 ACCAGCGAUA AUCCCUCGGU UUCAGACAGU GAGUAUGACC GCCUUUACCG UGAGUUGGUC 120 GAGUUAGAAA CUGCUUAUCC AGAGCAAGUG CUAGCAGACA GUCCGACUCA UCGUGUUGGU 180 GGCAAGGUUU UAGAUGGUUU UGAAAAAUAC AGUCAUCAGU AUCCUCUUUA UAGUUUGCAG 240 GAUGCUUUUU CACGUGAGGA GCUAGAUGCU UUUGAUGCGC GUGUUCGUAA GGAAGUGGCU 300 CAUCCGACCU AUAUUUGUGA GCUGAAAAUC GAUGGCUUAU CUAUCUCGCU GACUUAUGAA 360 AAGGGGAUUU UGGUUGCUGG GGUAACACGU GGAGAUGGUU CAAUUGGUGA AAAUAUCACA 420 GAAAACCUCA AGCGUGUUAA GGACAUCCCU UUGACUUUGC CAGAAGAACU AGAUAUCACA 480 GUUCGUGGGG AAUGUUACAU GCCACGCGCU UCCUUUGACC AAGUUAACCA AGCGCGCCAA 540 GAAAAUGGAG AGCCUGAAUU UGCUAAUCCU CGUAAUGCGG CAGCAGGAAC UCUGCGUCAG 600 UUGGAUACAG CAGUAGUUGC CAAGCGUAAU CUUGCAACGU UUCUCUAUCA AGAAGCCAGC 660 CCUUCAACUC GUGAUAGCCA AGAAAAGGGU UUGAAGUACC UAGAACAACU AGGUUUUGUG 720 GUCAAUCCUA AGCGAAUCUU GGCUGAAAAC AUAGAUGAAA UCUGGAAUUU UAUCCAAGAA 780 GUAGGACAGG AACGGGAAAA UCUGCCUUAC GAUAUUGAUG GAGUGGUAAU CAAGGUCAAC 840 GACCUAGCAA GUCAAGAAGA ACUUGGUUUU ACCGUUAAGG CUCCAAAGUG GGCAGUAGCC 900 UACAAGUUCC CUGCUGAAGA AAAAGAAGCU CAACUCUUAU CAGUUGACUG GACAGUUGGC 960 CGUACCGGUG UUGUAACUCC AACUGCUAAU CUAACACCAG UACAACUUGC CGGUACGACU 1020 GUUAGCCGUG CGACCCUGCA CAAUGUAGAU UAUAUUGCUG AAAAAGAUAU CCGAAAAGAC 1080 GAUACGGUCA UUGUAUAUAA GGCUGGUGAC AUCAUCCCUG CCGUUUUACG UGUGGUAGAG 1140 UCCAAACGGG UUUCUGAAGA AAAACUAGAU AUCCCUACAA ACUGUCCAAG UUGUAACUCU 1200 GACUUGUUGC ACUUUGAAGA UGAAGUGGCC CUACGUUGUA UCAAUCCGCG UUGCCCUGCU 1260 CAAAUCAUGG AAGGCUUGAU UCACUUUGCU UCUCGUGAUG CUAUGAAUAU UACAGGCCUU 1320 GGUCCAUCUA UUGUUGAGAA GCUUUUUGCU GCUAAUUUAG UCAAGGAUGU GGCGGAUAUU 1380 UAUCGUUUGC AAGAAGAGGA UUUCCUCCUU UUAGAGGGGG UUAAGGAAAA GUCCGCUGCU 1440 AAACUGUAUC AGGCUAUCCA AGCAUCAAAG GAAAAUUCUG CCGAGAAGCU CUUAUUUGGU 1500 UUGGGAAUUC GUCAUGUCGG AAGCAAGGCU AGUCAGCUUU UACUUCAAUA UUUCCAUUCA 1560 AUUGAAAAUC UGUAUCAGGC AGAUUCAGAG GAAGUGGCUA GUAUUGAAAG UCUAGGUGGC 1620 GUGAUUGCCA AAAGUCUUCA GACUUAUUUU GCGGCAGAAG GCUCUGAAAU UCUGCUCAGA 1680 GAAUUGAAAG AAACUGGGGU CAAUCUGGAC UAUAAAGGAC AGACGGUAGU AGCGGAUGCG 1740 GCCUUGUCAG GUUUGACCGU GGUAUUGACA GGAAAAUUGG AACGACUCAA GCGCUCAGAA 1800 GCUAAAAGUA AACUCGAAAG UCUGGGUGCC AAAGUGACAG GUAGUGUUUC UAAAAAGACC 1860 GACCUCGUCG UGGUAGGUGC AGACGCUGGA AGUAAACUGC AAAAAGCACA AGAACUUGGU 1920 AUCCAGGUCA GAGAUGAGGC AUGGCUAGAA AGUUUGUAA 1959 1557 base pairs nucleic acid single linear DNA (genomic) NO NO 4 TTTCAGCTCA CAAATATAGG TCGGATGAGC CACTTCCTTA CGAACACGCG CATCAAAAGC 60 ATCTAGCTCC TCACGTGAAA AAGCATCCTG CAAACTATAA AGAGGATACT GATGACTGTA 120 TTTTTCAAAA CCATCTAAAA CCTTGCCACC AACACGATGA GTCGGACTGT CTGCTAGCAC 180 TTGCTCTGGA TAAGCAGTTT CTAACTCGAC CAACTCACGG TAAAGGCGGT CATACTCACT 240 GTCTGAAACC GAGGGATTAT CGCTGGTATA GTACTCAGTC GCATAGCGAT TGAGCAAAGC 300 GACTAACTCA TTCATTCTTT TATTCATAAG ACCATTTTAC CATAAAACAA GCCCTCCTCA 360 CAAACGAGAA GGGCGGAAAA AACACTTAGT TTGAAATTAT TTTTGAAACT CAAGCAACCT 420 TATATCAATT TTTCAAAATG AGTTCGAACA TAAATAAACG ATATACAAGA CAAGATGATA 480 ACACCACTTC CAATTATCAG GAAAGAAGAG AGATGTACAC TTGGCAAGAC TGTCATAAAT 540 CCTTTTGCAA TAGGCATAAA TAGAATAGCT AAGGTAAAAA TTGTACTCAG TACTCTTCCA 600 AGAAATTCGC TCTCAACCTT GGTTTGTACT TGAGTAAAAA AGTGAATATT AAAAATCGTC 660 ATAAACAATT CACAAACTAA ATTTCCAGAA AAGGAAAGAA AAGTTGGAAG TGGTAATCCC 720 ATCATAAAAA CTCCGACACC TGTCAAAGCC AGTAAAATCA AAAGATTATA AATATTAGCT 780 TTAATTTTAC TAGCTAGAAG AGCCCCAATG ATGGAACCAA TAGCCCCCAT AGTTAAAATA 840 CTTGCATAGG CTCCTTCTGA CCCGTAAAGC TGATTCGAAA AGGGAAGTAG AAATTCAAAA 900 GCTGCAAAAA AGAAATTAAC GCTGGAAGCT ACCAGCAAAA GGAAGAAAAT TTCTTGCTGA 960 TGCCAGATAT AGTGTAACCC ATCCTTGATA TCTACAAAAA TATCTCTCCC AGTAAAAGCC 1020 TTTTTCTCTT GAACTTTTGC TTCCTCTTTT GGAAGGAAAG CCACTAGAAC AAAAGCAATG 1080 AAAAAAGTCA GCGAGTCTAG CAGTAGCGTC ATATGGAGAC TTGCAAACTG TAAAACAAGG 1140 AAGGAAAGAA CAGGAGAGCT AACACCTACA ACCTGCAAAA CCAGCTCTAA GCGAGAATTA 1200 TAGATCACAA TCTCATTTTT CTCCACCACT TCAGTTATGA TAGCTTTATT GGCTGTGCGA 1260 GAAAAGGCAA AAGCAATAGC CTGCACAATG TTAGCAACAA TCAAAGCGCC AATCATCCAG 1320 CTATCATTCC TTATGAAAGA AATAGCCAGA CAAAGAATCC CACAAACAAG ATCTGCCGTC 1380 ATTAAAATCT TACGACGAGA AAAACGGTCT GAAATAACTC CGCCAAAGGG ATTGACGAGA 1440 ATAGATGTGA CGAGCTCAGA AATCTGATAC ATTCCTAAAA CTGTCTGTCC TATAGTCCCC 1500 ATAGAAGCCA ACCAGACACT ATTTCCATAA TCATAGAGCA TATTCCCATT TTATTGA 1557 658 base pairs nucleic acid single linear DNA (genomic) NO NO 5 CTTATTTGGT TTGGGAATTC GTCATGTCGG AAGCAAGGCT AGTCAGCTTT TACTTCAATA 60 TTTCCATTCA ATTGAAAATC TGTATCAGGC AGATTCAGAG GAAGTGGCTA GTATTGAAAG 120 TCTAGGTGGC GTGATTGCCA AAAGTCTTCA GACTTATTTT GCGGCAGAAG GCTCTGAAAT 180 TCTGCTCAGA GAATTGAAAG AAACTGGGGT CAATCTGGAC TATAAAGGAC AGACGGTAGT 240 AGCGGATGCG GCCTTGTCAG GTTTGACCGT GGTATTGACA GGAAAATTGG AACGACTCAA 300 GCGCTCAGAA GCTAAAAGTA AACTCGAAAG TCTGGGTGCC AAAGTGACAG GTAGTGTTTC 360 TAAAAAGACC GACCTCGTCG TGGTAGGTGC AGACGCTGGA AGTAAACTGC AAAAAGCACA 420 AGAACTTGGT ATCCAGGTCA GAGATGAGGC ATGGCTAGAA AGTTTGTAAT GGATCGTTTA 480 AAAACAGAGT TTAGAGAATA TGACTATGTC TGTTAATTGA GACGAGATTG ACAAAAATTT 540 ATTAGTGAAA TAGGAAACAA AGTAAAAAGG AAAAATAAAA AATGTATACT ACCCTATGCG 600 CATTCATTAC CATCGTAAGA ATGGAGAATA TGACCTTGCT CCTTTGTAAA AGTCAGGA 658 

We claim:
 1. An isolated nucleic acid fragment, wherein said fragment has a sequence selected from the group consisting of: (a) SEQ ID NO: 1; (b) SEQ ID NO: 3; (c) a nucleic acid fragment that encodes the same protein as depicted in SEQ ID NO:2; and (d) a nucleic acid fragment fully complementary to (a), (b), or (c).
 2. The isolated nucleic acid fragment of claim 1, wherein the sequence of said fragment is selected from the group consisting of SEQ ID NO: 1, a nucleic acid fragment that encodes the same protein as depicted in SEQ ID NO:2 , and a nucleic acid fragment fully complementary to either of the foregoing.
 3. The isolated nucleic acid fragment of claim 1, wherein the sequence of said fragment is selected from the group consisting of SEQ ID NO:3, a nucleic acid fragment that encodes the same protein as depicted in SEQ ID NO:2 , and a nucleic acid fragment fully complementary to either of the foregoing.
 4. An isolated nucleic acid fragment encoding a protein having the amino acid sequence that is SEQ ID NO:2.
 5. A vector comprising the isolated nucleic acid fragment of claim
 4. 6. The vector of claim 5, wherein said isolated nucleic acid fragment is SEQ ID NO:1, operably-linked to a promoter sequence from said vector.
 7. A recombinant host cell containing said vector of claim
 6. 8. A recombinant host cell containing said vector of claim
 5. 9. A method for constructing a recombinant host cell having the potential to express a protein having the amino acid sequence shown in SEQ ID NO:2, said method comprising introducing into said host cell by any suitable means said vector of claim
 5. 10. A method for producing a protein having the amino acid sequence of SEQ ID NO:2 in said recombinant host cell of claim 7, said method comprising culturing the recombinant host cell under conditions suitable for production of said protein.
 11. The method of claim 10, further comprising recovering said protein.
 12. An isolated nucleic acid fragment, wherein said fragment consists essentially of a sequence selected from the group consisting of: (a) SEQ ID NO: 1; (b) SEQ ID NO: 3; (c) a nucleic acid fragment that encodes the same protein as depicted in SEQ ID NO:2; and (d) a nucleic acid fragment fully complementary to (a), (b), or (c).
 13. The isolated nucleic acid fragment of claim 12, wherein the sequence of said fragment is selected from the group consisting of: (a) SEQ ID NO:1; (b) a nucleic acid fragment that encodes the same protein as depicted in SEC ID NO:2 ; and (c) a nucleic acid fragment fully complementary to (a) or (b).
 14. The isolated nucleic acid fragment of claim 12, wherein the sequence of said fragment is selected from the group consisting of: (a) SEQ ID NO:3; (b) a nucleic acid fragment that encodes the same protein as depicted in SEQ ID NO:2 ; and (c) a nucleic acid fragment fully complementary to (a) or (b).
 15. An isolated nucleic acid fragment consisting essentially of a nucleotide sequence encoding a protein having the amino acid sequence that is SEQ ID NO:2.
 16. A vector comprising said isolated nucleic acid fragment of claim
 15. 17. The vector of claim 16, wherein said isolated nucleic acid fragment is SEQ ID NO:1, operably linked to a promoter sequence from said vector.
 18. A recombinant host cell containing said vector of claim
 17. 19. A recombinant host cell containing said vector of claim
 16. 20. A method for constructing a recombinant host cell having the potential to express a protein having the amino acid sequence shown in SEQ ID NO:2, said method comprising introducing into said host cell by any suitable means said vector of claim
 16. 21. A method for producing a protein having the amino acid sequence of SEQ ID NO:2 in the recombinant host cell of claim 18, said method comprising culturing said recombinant host cell under conditions suitable for production of said protein.
 22. The method of claim 21, further comprising recovering said protein. 